Coding

Part:BBa_K5332003:Design

Designed by: Qilin Yu   Group: iGEM24_NKU-China   (2024-09-26)


CMC (arttificial adhesion protein)


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 470
    Illegal PstI site found at 673
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1444
    Illegal PstI site found at 470
    Illegal PstI site found at 673
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 470
    Illegal PstI site found at 673
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 470
    Illegal PstI site found at 673
    Illegal NgoMIV site found at 381
    Illegal NgoMIV site found at 1806
    Illegal NgoMIV site found at 1810
    Illegal AgeI site found at 295
    Illegal AgeI site found at 1396
  • 1000
    COMPATIBLE WITH RFC[1000]

Design Notes

We contributed an innovative foundational component to the Registry platform: the artificial glucan-binding protein CMC, designated as BBa_K5332002. This component ingeniously integrates the mechanism of glucan-binding proteins with the scaffold protein CipC from Clostridium cellulovorans. Additionally, we incorporated the mCherry fluorescent protein sequence and the outer membrane protein A (OmpA) signal peptide, designing proteins with multiple copy numbers to optimize performance. To enhance the persistence of engineered strain FMK in the gut and its response in expressing anti-inflammatory factors, we designed a novel artificial glucan-binding protein CMC by simulating the natural mechanisms of glucan-binding proteins. CMC acts as a "bridge" between gut microbiota and the intestinal surface, effectively attracting and recruiting beneficial gut probiotics. This design not only promotes stable adherence of the engineered strain in the gut but also significantly enhances its efficacy in alleviating intestinal inflammation and modulating the gut microenvironment. radhesion1-1.png

To ensure the effective and sustained retention of engineered bacteria in the gut, and their responsive expression of anti-inflammatory factors, we identified this as a key to enhancing therapeutic efficacy. After reviewing extensive literature, we found that the main component of intestinal mucus is the highly glycosylated glycoprotein MUC2, which contains various glycan structures such as Core1, Core2, and Core3. Additionally, glucans are important polysaccharides produced by bacteria and fungi. The beneficial properties of probiotics are often related to their production of extracellular polysaccharides (EPS), with many probiotics exposing glucans on their surfaces, such as the α-glucans of Lactobacillus. Some pathogenic bacteria, like Salmonella, have cell walls containing endotoxin lipopolysaccharides (LPS) with O antigens.

We realized that leveraging glucan-binding properties could lead to the design of an adhesion factor that acts as a "bridge" between gut microbiota and the intestinal surface, thereby stabilizing the attachment of engineered bacteria in the gut. Inspired by the binding of glucan-binding proteins to glucan substrates, we designed the CBMCipC domain. CBMCipC, derived from the scaffold protein CipC of *Clostridium cellulovorans*, includes a type III cellulose-binding domain (CBD), a hydrophilic domain, and two hydrophobic domains. The CBD domain endows CBMCipC with glucan-binding capability.

Source

The core component of CMC, CBMCipC, is derived from the scaffold protein CipC of Clostridium cellulolyticum

References

1 NIE Shuo, WEN Zhengshun. Secretion, Structure, Synthesis Regulation of Intestinal Mucin 2 and Its Role in Development of Intestinal Diseases. Chinese Journal of Animal Nutrition, 2020, 32(6): 2521-2532.

2 Pourjafar, Hadi et al. “Functional and health-promoting properties of probiotics' exopolysaccharides; isolation, characterization, and applications in the food industry.” Critical reviews in food science and nutrition vol. 63,26 (2023): 8194-8225.

3 Yu, Liansheng et al. “Glucansucrase Produced by Lactic Acid Bacteria: Structure, Properties, and Applications.” Fermentation (2022): n. pag.

4 Chen, Ziwei et al. “Lactic acid bacteria-derived α-glucans: From enzymatic synthesis to miscellaneous applications.” Biotechnology advances vol. 47 (2021): 107708.

5 Fabrega A., Vila J. (2013). Salmonella enterica serovar Typhimurium skills to succeed in the host: virulence and regulation. Clin. Microbiol. Rev. 26 308–341. 10.1128/CMR.00066-12

6 Whitfield, Chris et al. “Lipopolysaccharide O-antigens-bacterial glycans made to measure.” The Journal of biological chemistry vol. 295,31 (2020): 10593-10609.

7 Branchu, Priscilla et al. “Genome Variation and Molecular Epidemiology of Salmonella enterica Serovar Typhimurium Pathovariants.” Infection and immunity vol. 86,8 e00079-18. 23 Jul. 2018

8 Pages, S., Gal, L., Belaich, A., Gaudin, C., Tardif, C., Belaich, J.P., 1997. Role of scaffolding protein CipC of Clostridium cellulolyticum in cellulose degradation. J. Bacteriol. 179, 2810–2816.

9 Park, Jeong Soon et al. “Mechanism of anchoring of OmpA protein to the cell wall peptidoglycan of the gram‐negative bacterial outer membrane.” The FASEB Journal 26 (2012): 219 - 228.

10 Yin, Hongda et al. “Synthetic physical contact-remodeled rhizosphere microbiome for enhanced phytoremediation.” Journal of hazardous materials vol. 433 (2022): 128828.